Effects of mesotrione on the control efficiency and chlorophyll fluorescence parameters of Chenopodium album under simulated rainfall conditions

This experiment was conducted to study the effects of mesotrione on the control efficiency and chlorophyll fluorescence parameters of Chenopodium album. Simulating three rainfall intensities of 2 mm/h (light rain), 6 mm/h (moderate rain) and 10 mm/h (heavy rain) at different interval times (0.5 h, 1 h, 2 h, 4 h) to analyze variable regulation of the control effect, the photosynthetic pigment content and chlorophyll fluorescence parameters of C. album after spraying mesotrione. With the extension of rainfall time interval, the inhibition rate of plant height, plant control effect and fresh weight control effect of C. album were gradually increased, the inhibition effect of rainfall on the efficacy was gradually decreased, at the same time, the contents of chlorophyll a, chlorophyll b, carotenoids, the maximum photochemical quantum efficiency (Fv/Fm), the actual photochemical quantum yield (Y (II)) and quantum yield (Y (NO)) production of regular energy consumption of C. album were also increased, while the nonregulatory energy decreased gradually. The results showed that the contents of chlorophyll a and chlorophyll b in leaves of C. album increased significantly by 35.63% and 35.38% compared with the control under the condition of simulating 6 mm/h in interval 1 hour. The study suggested that simulating 10 mm/h rainfall intensity had the greatest effect on C. album, the photosynthetic pigment content, Fv/Fm and Y (II) of leaves were significantly higher than those in the control groups under 0.5 h, 1 h and 2 h interval treatments. The carotenoid content was the lowest and Y (NO) was the largest under the 4 h interval treatment. As is displayed that rainfall reduced the weed control effect in the aspect of controlling C. album on mesotrione, which is partly contributed to increase photosynthetic pigment content and enhance the PS II photochemical activity. In conclusion, the rain intensity of ≤2 mm/h did not affect the control effect of mesotrione on C. album. At 6 mm/h within 1 h after treatment, the control effect of fresh weight was significantly reduced by more than 7.14%, and at 10 mm/h within 2 h, the control effect was significantly reduced by more than 14.78%.

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• * typeset Additional data availability information: 1 Introduction 52 Weed would reduce the yield and quality of the crops, hinder mechanized sowing and harvest 53 and then slow down the process of agricultural modernization [1][2]. Chenopodium album. L is an 54 erect annual herbaceous plant, that grows between 0.2 and 2 meters in height, striped green, red or 55 purple stems. Leaves are rhomboid, deltoid to lanceolate, upper entire, lower toothed or irregularly 56 lobed. The leaves are wax-coated, sometimes has mealy and unregular sawtooth on the edge of 57 them, with a whitish coat on the underside. The length of its petiole is close to blade or half of the 58 blade [3]. C. album is one of the common farmland malignant weed in northern China. It has strong 59 environmental adaptability and a large root system, even grow on the Tibetan Plateau and in the 60 lowlands of southwest China, so the C. album is recorded as a potherb in both plateaus and low 61 lands [4]. It has obvious advantages when competing with crops for water and fertilizer, every 62 single plant could yield 3000-20000 seeds (Data from CABI), which led to a sharp decline in crop 63 yield [5-6]. Sugarbeet, barley, mustard, gram, maize and so many crops were defeated by C. album, 64 it has been reported to reduce the soybean (Glycine max) yield by 61%, in the wheat (Triticum 65 aestivum) field, the yield loss approximately 50-60%, which also was associated with interference 66 of this weed [7][8][9]. When the density increases to 20 plants per m 2 , the corn would not ear because 67 of the shade effect of C. album [10]. 68 Mesotrione is a herbicide of broad-spectrum, selective stem and leaf before bud and after 69 seedling mainly used to effectively control broad-leaved weeds and some gramineous weeds by 70 inhibiting the catalytic factors of plant photosynthetic process. Mesotrione is widely used in weed 71 control of maize and winter wheat due to its high activity, low residue, strong compatibility and 72 safety to the environment and subsequent crops [11][12]. Summer is the key period for crop growth 73 and weeding. However, local microclimate and severe convective weather occur frequently, and 74 rainfall interfere the herbicide efficacy due to the scouring every year [13]. Therefore, it is attached 75 importance to study the influence of rainfall on herbicides. There are great differences in the 76 absorption rate and rain resistance among different stem and leaf treatment agents, so the interval 77 time of affecting the efficacy is different, but most herbicides will not affect the weed when rainfall 78 after 4-6 h [14]. Wu et al. [15] found that rainfall after the application had a significant inhibitory 79 effect on the efficacy of nicosulfuron methyl; paraquat could effectively control Alopecurus 80 aequalis after 0.5 h and 1 h rainfall [16]; there was no significant difference in bensulfuron efficacy 81 between without rainfall and after 2 h rainfall [17]; Wang et al. [18] showed that the effect of 82 imazethapyr was no longer affected under the 9 mm/h rainfall intensity. When the interval time of 83 rainfall is 0.5 h, the effect of rainfall on barnyardgrass was significantly reduced, it's down to 19.42%

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[19]. At present, it is known that mesotrione has effects on the growth of weeds and crops, such as 85 reducing the plant height of maize, making Amaranthus retroflexus heart leaves be yellow, reducing of mesotrione on broad-leaved weed C. album under rainfall conditions. In this experiment, 94 artificial rainfall was used to simulate rainfall, and C. album is a common malignant weed in the 95 field, was used to simulate different rain intensities at different intervals after spraying mesotrione.

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In order to assess the possibility of mesotrione application and understand the related 97 mechanism, we investigated the weed control, agronomic traits, photosynthetic pigment content and 98 5 the chlorophyll fluorescence parameters of C. album to mesotrione in this current study. The 99 influence of rain intensity and interval time on the efficacy was determined, which provide a 100 theoretical basis for the use of mesotrione. and belongs to a warm temperate continental monsoon climatic with an altitude of approximately 106 800 meters, the annual average temperature was 9.9 ℃, and the frost-free period was about 159 107 days. Annual precipitation of the province was 400-650 mm, but the seasonal distribution was 108 uneven, with more than 60% of precipitation concentrated in June to August [24]. Tables 1 and 2 109 present the meteorological conditions and soil characteristics of experimental site.  The chlorophyll fluorescence parameters were tested at 12 h, 24 h, 2 d and 4 d after application   inhibited, the tip of leaves was more serious, and the heart leave was even completely withered and 180 shed. The survey of weed injury level ( 8.43%, 6.80%, respectively) when the rain intensity is 10 mm/h, and the reduced efficiency was 208 above 7%.

Effects of simulated rainfall on photosynthetic pigment content of C.
212 album leaves after spraying mesotrione The data in Figure 1 showed that the corresponding rainfall intensity was 2 mm/h, compared 216 with CK at 3 DAT, T1 and T2 treatments show a significant increase, 24.73% and 17.2%. Compared 217 with all interval treatments, CK at 6 DAT did not see a big difference, which could indicate that the 218 rainfall in this period is not capable of preventing the albino process of C. album.

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When the rainfall intensity reached 6 mm/h, it could find that the corresponding carotenoid 220 content of T1 and T2 treatments increased at 3 and 6 DAT, and the ranges were 21.85%, 13.89%, 221 20.00% and 21.91%, respectively. Except that the T4 interval treatment was a bit different, the 222 carotenoid content involved in other treatments looked higher than the content contained in CK.

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And the growth rates are 39.78%, 36.56%, 29.03% and 33.32%, 19.32%, 15.79%.  and showed a decreasing trend with the extension of rainfall interval. As shown in there was no significant difference between T1, T2 and T3 treatment. 15

Effects of simulated rainfall on chlorophyll fluorescence
254 parameters of C. album after spraying mesotrione In addition, it can be seen that there is no big difference between T4 interval treatment Y(II) 265 and CK, and the remaining treatment Y(II) has a greatly increasment , whose amplitude are 43.85%,

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Compared to the situation of CK, Y(II) of C. album had a increasment by 528.50% and 398.96% 296 at 4 DAT for T1, and T2 treatments. And it is noted that it is almost the same as T3, T4 treatments 297 and CK (Figure 3. B), stating that if the coppresponding rain intensity reached 6 mm/h, the rainfall 298 within 1 hour could be give a great influence at the efficacy of mesotrione.

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The Y(NPQ) value of C. album was measured at different periods, and its magnitude was as 300 follows: 24 hours after treatment>12 hours after treatment>2 d after treatment>4 d after treatment.

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The photosynthetic inhibitory effect of C. album was the strongest at 4 DAT. Compared with 12 302 hours after application, because of the self-protection mechanism of C. album, Y(NPQ) will has a 303 slower increasment, and then slowly decreased; Y(NO) showed that with the extension of the   intensified, the symptoms are more significant later. Spillman [39] think the rainfall would dilute or 359 wash the medicament on weed's surface, and then the reduced efficacy couldn't have a thorough 360 effect on the weed. In this study, on condition that the rain intensity was 2 mm/h and the rainfall 361 interval was T1, the plant height inhibition rate of C. album was still close to that of no rainfall after 362 application, indicating that a light rain after application would not affect the efficacy of mesotrione.

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When the rain intensity is 6 mm/h, under the treatment of 2 h interval, with the extension of the rain 364 interval time, the control effect of mesotrione on C. album gradually increases, and the control 365 effect of plant and fresh weight is still above 90%, it only decreased by 0.4%-2.47% compared with 366 CK, the efficacy of mesotrione was not significantly affected.

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The mechanism of action of herbicides is mainly to cause weed death by interfering or have better wettability, adhesiveness, penetrating quality and resistance to rain washing capability 416 and ultimately the efficacy would be increased [51]. Tao et.al [52] researched that adjuvants could 417 increase the prevention and control effect if the rainfall appears in 4 h after mesotrione was applied.

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Terrible environment would reduce herbicide's efficacy, but effective ingredient in the adjuvants 419 could increase herbicide's prevention and control effect by counteracting the influence. And how to 420 choose adjuvants needs to be further studied.  Authors' contribution 434 The author would like to thank all authors for their help in the experience and manuscript.       Chenopodium is the main research object of our experiment, so we didn't collect other weed species.

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The plant was weighed after washing, the biomass of each weed sample were measured. The     The data in Figure 1 showed that the corresponding rainfall intensity was 2 mm/h.        hours, the effect of mesotrione will be greatly reduced due to rainfall. simulation. Firstly, we screened out one type of shower that sprayed the smallest water and most homogeneous when spraying. We also controlled the moving speed of spray lance by ourselves and found it is equal to the rainfall whose intensity is 2、6、10 mm/h when the capable of spraying was 4, 12 and 20 L per half an hour. According to the means and data of the preliminary experiment, we chose a windless and sunny day in the field experiment, and we put many baffles around the rainfall simulation areas of the neighborhood like the preliminary experiment. The simple facilities that put up by us when we test the rainfall intensity are as follows.

Response to comment:
The experiment was conducted for one season only (4 months), whereas repeated results can explain the cause and effects better.

Response:
The experiment was mainly conducted in the field, and we tested it again in the greenhouse. Ultimately, the conclusion we drew in the field and the greenhouse were almost consistent. These are some of the photos from our greenhouse experiments. Response: Before the start of the field test, we conduct an experiment for two months when the intensity and the precipitation time were suitable. The concrete operations are as follows： when spraying and mark the appropriate place in the electric spray knob by many tests, screening out one type of shower that sprayed the smallest water and most homogeneous, and we moved the spray lance in a constant speed within the unit area. The electric sprayer was continuously adjusted to the appropriate site according to the water yield, so that the water yield of 0.5 h was 4 L, 12 L and 20 L, reaching the rain intensity standard of 2, 6 and 10mm/h.
The error is maintained at ±0.2 mm/h. The height of the spray is 0.5 meters, mark the wall in advance, such as the position of the green line in the last picture. In the newly submitted manuscript, we have rewritten the Materials and methods.
11．Response to comment: Only Chenopodium plans were measured? What about remaining weed species?
Response: Chenopodium is easy to survive under any bad existent conditions. It competes with crops on the water and nutrition, so the yield and the quality of crops sharply drop to a low level.
And then the agriculture suffers massive loss. The damage that is caused by Chenopodium interference has been well documented in field crops. Chenopodium has been reported to reduce the barley (Hordeum vulgare L.) yield by 36% [1] . In sugarbeet (Beta vulgaris L.) field, the yield was reduced by 48%, which was associated with interference of this weed [2] . The presence of Chenopodium in corn fields has been shown to reduce the yield by 58%, and the yield of soybean will drop by 61% in the severe cases [3] . Chenopodium are always companied by the mixed agriculture pattern of millet (both foxtail millet and broomcorn millet), rice and wheat [4] . The experiment was carried out in foxtail millet field, Chenopodium is one of major weeds, so we did this experiment that focused on the Chenopodium. Of course, other weeds can also be targeted for future research, we will continue our study, thanks for your advice! caused in your reading. The manuscript has been thoroughly revised.
17. Response to comment: Results are clearly explained but, Herbicide effect was studied on weed under sole weed cultivation.
Response: Chenopodium is listed among the most common dicotyledonous weeds in the world and it's widely distributed in many agricultural areas, which severely reduce the yield and decrease the quality of the crops [1] . Because Chenopodium is one of the main weeds in the foxtail millet field and it is flooding in the drought areas, so we choose Chenopodium as our primary research object [2] . In order to show that the efficacy of mesotrione to weeds under different intensity, we use Chenopodium as the only research object in the experiment. We can regard the other weeds as a research in the following experiment period.
Reference: Response: We understand that combined crop plant stand would better. However, my senior schoolmates who study in our laboratory, he did some research about how much mesotrione would be safe or harmful for the foxtail millet. He found that the mesotrione is suitable for the foxtail millet field under the dose about 75-300 g/hm 2 . Because the weather in summer is changeable and rainy days are common here, so we had a further experiment to study if the rainwash would have an effect on the mesotrione and what effect would have. Then we can hope to screen out a safe and effective herbicide. In the present study, we mainly focused on the rainwash of the mesotrione and regarded Chenopodium as an assistant research object. We think that study under sole weed may not be optimal, but should be sufficient to draw my conclusion about the effect of rainfall intensity on mesotrione. As for the influence in the mixed condition, we will make further in-depth study, many thanks for your kind help! 19. Response to comment: No data on Chenopodium leaf area, since LA is the most important factor decides the efficacy of herbicide.
Response: Thank you for your instructive suggestions. We are sorry for not describing the data on Chenopodium leaf area in our manuscript. All leaf area were conducted statistical analyses by Image J, we found that the leaf area of the treatment was between 26.7 to 39.4 cm 2 . When the rain intensity was 2 mm/h, the plant height inhibition rate, plant control efficiency and fresh weight control efficiency of Chenopodium at intervals of 0.5 h and 4 h decreased by 18.07%, 18.12%, 16.25%, 4.86%, 1.50% and 2.01% respectively at 15 DAT. So we can infer that the loss of the herbicide is between 1.50% and 18.12%. In the same way, the weed control loss of the herbiside between 16.02% and 48.25%. when the rainfall intensity is 6 mm/h. And the loss is between 17.38% and 52.82% when the rainfall intensity is 10 mm/h, and we have added these datas in the new manuscript. As for the detail about herbicide loss, we will make a further study.

Response to comment:
Thanks for your meaningful comments! 22. Response to comment: Discussion is fine with the supporting literature. However, your results could have discussed more, for example time of rain interval, all rain intensities and interaction effect, what extent it affected.
Response: Your comments were highly insightful and enabled us to greatly improve the quality of our manuscript. We have re-written Discussion according to the Reviewer's suggestion.
We hope that the correction will meet with approval, thank you very much for your comments and suggestions.

Response to comment:
Discussion section is lacking the supporting data on herbicide retention on leaves, leaching loss, biomass reduction.
Response: I agree with you ! Herbicide retention on leaves, leaching loss, biomass reduction are important datas what could show the efficacy of the mesotrione straightly. We only explore the control effect of mesotrione and the physiological indexes on Chenopodium under different rainfall intensity, this is a pretty basical research. We have konwn the loss and the residual of the herbiside are closely related to the efficacy. Hence, our group is going to carry out further experiments.
In addition to the above commernts, there is a comment regarding data in Table 3.
1. Response to comment: I think treatments comparison was done for T1 to T4 within the rainfall intensities. Whereas, comparison between all the treatment combinations (3*5= 15) would be good.
Response: I also think comparison between all the treatment combinations (3*5= 15) is a good idea. But in our experiment, we could intuitively observe that the efficacy loss of the mesotrione will be serious when the rainfall intensity was large. And the emphasis of this experiment was comparing the effect of rainfall interval to the efficacy of mesotrione under different rainfall intensity. Hence in order to draw a nice conclusion, we compared them separately.
We tried our best to improve the manuscript and made some changes in the manuscript.
These changes will not influence the content and framework of the paper. And here we did not list the changes but marked in red in revised paper. We appreciate for Reviewers' warm work earnestly and hope that the correction will meet with approval. Once again thank you very much for your comments and suggestions.
In all, I found the reviewer's comments are quite helpful, and I revised my paper point-by-point. Thank you and the review again for your help.